Mycoplasma genitalium detection assay based on the mg219 gene

ABSTRACT

There is provided a method for detecting  M. genitalium  nucleic acid in a sample, comprising: (i) amplifying a nucleic acid sequence comprising a fragment of SEQ ID NO: 1 (Mg219 gene); and (ii) detecting said amplified nucleic acid sequence.

The present invention relates to detection of Mycoplasma sp., inparticular, Mycoplasma genitalium, and to reagents and kits therefor.

The Class Mollicutes contains Gram-positive bacteria that lack a cellwall, including the genera Mycoplasma, Acholeplasma and Ureaplasma.There are over 100 species of Mycoplasma alone, and 17 species ofMollicutes are considered a part of the human flora—including M.pneumoniae, M. genitalium, M. fermentans and M. penetrans.

Human and mammalian Mycoplasmas infect cells comprising mucus membranesand can have strict host and tissue specificities. By way of example,the primary tissue infected by M. pneumoniae is the respiratory tract,whereas M. genitalium primarily infects the urogenital tract.

M. genitalium is sexually transmissible, with a transmission rate thatis believed to be similar to Chlamydia trachomatis. Indeed, cliniciansworking in the field have described M. genitalium as “the nextChlamydia”. M. genitalium is a causative agent in urogenital tractdiseases, in particular non-gonococcal urethritis (NGU). M. genitaliumhas also been implicated in pelvic inflammatory disease (PID) andinfertility in women. Extragenital infections with M. genitalium mayalso occur, for example in the respiratory tract, in the eye, and insynovial fluid—leading to sexually acquired reactive arthritis (SARA).

Antibiotics used for the treatment of mycoplasmal infections belong totetracyclines, macrolides-lincosamides and fluoroquinolones. Theseproducts are highly active in vitro against Mycoplasmas. However, someof these antibiotics have a differential activity according to species,and acquired resistance has been reported, mainly in genitalMycoplasmas. By way of example, M. genitalium is resistant tochloramphenicol. Most mycoplasmal infections are treated using adaptedantibiotics, but they may be difficult to treat in immunosuppressedpatients.

Mycoplasmas such as M. genitalium are very difficult to study byclassical genetic methods, both because of their fastidious growthrequirements and as a consequence of the absence of selectable markers.As a consequence, isolates have been very difficult to obtain.

Furthermore, the antigenic relationship between M. pneumoniae and M.genitalium leads to cross-reactions, which significantly hamper the useof serology for diagnostic and epidemiological studies.

Hybridization based techniques for detection of Mycoplasmas include theuse of DNA probes. By way of example, radiolabelled oligonucleotideprobes have been described that target the M. genitalium 16S rRNA.

Because of the problems encountered using traditional procedures for thediagnosis of M. genitalium infection, such as culture and serology,knowledge about its pathogenicity has been slow to accrue. Extensiveclinical studies have only become possible with the advent of PCR-basedassays.

PCR has proven a particularly useful tool for detecting fastidiousMycoplasmas due to its very high sensitivity (Jensen, Dan. Med. Bull.2006; 53: 1-27). However, until recently, a major drawback of thistechnique has been the lack of commercial kits.

PCR assays have recently been developed that amplify and detect the M.genitalium MgPa gene (e.g. Jensen et al., J. Clinical Microbiology,February 2004, pages 683-692). The MgPa gene encodes the major M.genitalium adhesion protein responsible for attachment to host cells, inparticular for attachment to the epithelial cells, such as those of thehuman fallopian tube.

Evasion of the host immune system by antigenic variation of surfacecomponents such as adhesins enables Mycoplasma such as M. genitalium toadapt to changing environments and selection pressures. This antigenicvariation leads to considerable sequence variation in genes that encodesurface proteins such as the MgPa gene. Thus, diagnostic assays thatrely on detection of surface proteins and corresponding nucleic acidsfail to detect some variants, resulting in poor sensitivity of theassay.

Other published PCR-based assays for detecting M. genitalium aredirected towards the 16S rRNA gene (Yoshida et al., J. Clin. Microbiol.2002: 40:1451-1455). However, the specificity and sensitivity of thisassay has been put into question due to the high level of homologybetween the M. pneumoniae and M. genitalium 16S rRNA genes, and thepreponderance of secondary structures in the 16SrRNA.

A LightCycler assay has also been described, directed towards detectionof the P115 (MG299) gene with locked nucleic acid probes (Dupin et al.,Clin. Infect. Dis. 2003; 37:602-605).

There is, therefore, a need to provide an alternative and/or improvedsystem for detecting M. genitalium.

According to a first aspect, the present invention provides a method fordetecting M. genitalium nucleic acid in a sample, comprising: (i)amplifying a nucleic acid sequence comprising SEQ ID NO: 1 or a fragmentthereof; and (ii) detecting said amplified nucleic acid sequence.

According to a second aspect, the present invention provides a methodfor detecting M. genitalium nucleic acid in a sample, comprising: (i)contacting said sample with a probe, wherein the probe binds to a targetsequence within SEQ ID NO: 1, or the complement thereof; and (ii)detecting binding of said probe to said target site.

The present invention provides improved specificity and sensitivitycompared to existing PCR-based tests for M. genitalium.

The M. genitalium Mg219 gene (SEQ ID NO: 1) is 447 nucleotides long andencodes a 148 amino acid polypeptide of unknown function (SEQ ID NO: 2).

The Mg219 gene (SEQ ID NO: 1) is located from nucleotide residue 265596to nucleotide residue 266042 of the complete genome of M. genitalium (asdetailed in Accession No. NC 000908, M. genitalium G-37).

Mg219 is found within an operon consisting of the Mg217, Mg218 and Mg219genes, and transcription of Mg219 is continuous from Mg217 and Mg218(Musatovova O. et al., FEMS Microbiol. Lett., 2003, 5, 229(1) pages73-81). Mg218 encodes a high molecular mass cytadherence-related proteinrequired for tip-mediated adherence. The function of Mg219 remainsunknown, but due to the proximity to Mg218 in the M. genitalium genome,it is possible that Mg219 may be involved in adherence.

A detailed review by the present Applicant of all published Mycoplasmawhole genome sequences revealed that the Mg219 gene has no homologues inMycoplasma species other than M. genitalium.

Furthermore, by searching published genetic and protein databases, thepresent Applicant was unable to identify any gene having significanthomology to the Mg219 gene, or any protein having significant homologyto the encoded Mg219 polypeptide.

These findings were confirmed using the ORFANGE web-based program, whichidentifies genes that are only found once in a bacterial genome and notin other genomes.

Thus, the present Applicant has unexpectedly identified that the Mg219gene is specific to M. genitalium, and is thus useful for identificationof M. genitalium in a sample.

It is particularly important to keep the incidence of “false positive”results as low as possible in the field of detecting and diagnosingsexually transmitted diseases, due to the sensitive nature of the testresults for the patient, cost and implications of inappropriatetreatment, and the legal implications of providing an incorrect result.

Advantageously, because the Mg219 gene has no sequence homologues inother Mycoplasma species, or in other published sequence databases, thedetection assay of the present invention, which is based on detection ofMg219 nucleic acid sequences, is very specific, with a very lowincidence of “false positive” results.

As illustrated in the Examples (below), when a detection assay accordingto one embodiment of the present invention was tested by the presentApplicant against DNA from all known human Mollicutes, some otherbacterial species and pathogenic micro-organisms, the assay onlydetected M. genitalium thus illustrating the high degree of specificityof the assay.

The target Mg219 gene detected in the present invention is also anadvantageous target gene because it is conserved between differentstrains of M. genitalium. Thus, the assay of the present inventionadvantageously detects all known strains of M. genitalium that have beentested in the assay to date.

A sample may be for instance, a food, sewerage, environmental,veterinary or clinical sample. In one aspect, the method may be used fordetection of M. genitalium in a clinical sample.

Clinical samples may include urethral swabs, vaginal swabs, cervicalswabs, rectal swabs, penile swabs, throat/oral swabs, urine, blood,respiratory tract samples, synovial fluids, cerebro-spinal fluid, liquidbased cytology samples, tissue biopsies, ulcer samples, conjunctivitissamples and any other samples from animals, particularly from humans. Inone aspect, the sample may comprise semen or eggs.

Thus, in one aspect, the method of the present invention comprises thestep of amplifying M. genitalium nucleic acid.

In this application, the expressions “amplified nucleic acid sequence”and “amplicon” are used interchangeably and have the same meaning.

Specifically, in one aspect, the method of the present inventioncomprises amplifying a nucleic acid sequence comprising SEQ ID NO: 1.The amplified nucleic acid sequence (amplicon) may consist of SEQ ID NO:1.

In one aspect, the method of the present invention comprises amplifyinga nucleic acid sequence comprising a fragment of SEQ ID NO: 1. Theamplified nucleic acid sequence (amplicon) may consist of a fragment ofSEQ ID NO: 1.

Alternatively, the amplicon may comprise a fragment of SEQ ID NO: 1 andalso 1 or more additional nucleotides that are located upstream ordownstream of SEQ ID NO: 1 in the M. genitalium genome.

A fragment of SEQ ID NO: 1 is preferably at least 10 consecutivenucleotides of SEQ ID NO: 1, and is more preferably at least 25nucleotides, more preferably at least 50 nucleotides, and may be atleast 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375 or400 nucleotides of SEQ ID NO: 1. A fragment of SEQ ID NO: 1 ispreferably up to 440 consecutive nucleotides of SEQ ID NO: 1, morepreferably up to 425 nucleotides, more preferably up to 400 nucleotides,and may be up to 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125,100, 75, 50, or 25 nucleotides of SEQ ID NO: 1.

In a specific example, the amplicon may comprise a stretch ofconsecutive nucleotides spanning the nucleotide located 11 positionsupstream of SEQ ID NO: 1 in the M. genitalium genome (i.e. nucleotideposition −11) to the nucleotide at position 240 of SEQ ID NO: 1. Thus,in this specific example, the amplicon comprises a 240 nucleotidefragment of SEQ ID NO: 1, from nucleotide 1 of SEQ ID NO: 1 tonucleotide 240 of SEQ ID NO: 1 (plus the 11 nucleotides upstream of SEQID NO: 1 in the M. genitalium genome).

Amplification may be carried out by methods known in the art, preferablyby PCR. Examples of PCR platforms suitable for conducting theamplification step of the present invention include real-time platformssuch as Rotor-gene, LightCycler and Taqman platforms.

In one aspect, amplification of M. genitalium nucleic acid is carriedout using a pair of sequence specific oligonucleotide primers, whereinsaid primers bind to target sites in the M. genitalium nucleic acid.Under suitable conditions, the primers are extended, resulting innucleic acid synthesis. A skilled person would be able to determinesuitable conditions for promoting amplification of a nucleic acidsequence comprising a fragment of SEQ ID NO: 1.

For the avoidance of doubt, in the context of the present invention, thedefinition of an oligonucleotide primer does not include the full lengthMg219 gene (or complement thereof).

Primers of the present invention are designed to bind to the target genesequence based on the selection of desired parameters, usingconventional software, such as Primer Express (Applied Biosystems). Inthis regard, it is preferred that the binding conditions are such that ahigh level of specificity is provided. The melting temperature (Tm) ofthe primers is preferably in excess of 50° C. and is most preferablyabout 60° C. A primer of the present invention preferably binds totarget M. genitalium nucleic acid but is preferably screened to minimiseself-complementarity and dimer formation (primer-to-primer binding).

The primer pair comprises forward and reverse oligonucleotide primers.

A forward primer binds to the complementary (i.e. anti-sense) strand ofthe target M. genitalium nucleic acid and a reverse primer binds to thecoding (sense) strand of the target M. genitalium nucleic acid.

The forward and reverse oligonucleotide primers are typically at least 5nucleotides long, preferably at least 10 nucleotides long, morepreferably at least 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides long.Preferably, the primers are up to 60 nucleotides long, preferably up to55 nucleotides long, more preferably up to 50, 45, 40, 35, 30, 25, 20,15 or 10 nucleotides long.

In one aspect, the forward primer and/or the reverse primer is about50-55 nucleotides long. In one aspect, the forward primer and/or thereverse primer is about 20-25 nucleotides long. In one aspect, theforward primer and/or the reverse primer is about 25-30 nucleotideslong. In one aspect, the forward primer is about 29 nucleotides long andthe reverse primer is about 27 nucleotides long. It is an advantage touse shorter primers, as this enables faster annealing to target nucleicacid.

The forward primer binds to a target site within the M. genitaliumnucleic acid. This target site may be located within the complement ofSEQ ID NO: 1—i.e. the target site may be located between nucleotide 1and nucleotide 447 of the complement of SEQ ID NO: 1.

Reference to “a target site between (for example) nucleotide residues 1and 447 of the complement of SEQ ID NO: 1” does not imply that thetarget site extends between or is defined by the recited residues. Thus,the target site for the forward primer may be 447 nucleotides long, ormay be shorter, even considerably shorter than 447 nucleotides in length(e.g. up to about 200, 100, 75, 60, 50, 40, 30, 25, 20, 15, 10nucleotides long), so long as it is located somewhere within the recitedrange of nucleotide residues.

In one aspect, the target site of the forward primer is about 50-55nucleotides long. In one aspect, the target site of the forward primeris about 25-30 nucleotides long, preferably about 29 nucleotides long.

Alternatively, the target site for the forward primer within the M.genitalium nucleic acid may be located within upstream nucleic acidsequences that flank the complement of SEQ ID NO: 1 in the M. genitaliumgenome.

SEQ ID NO: 1 is located from nucleotide residue 265596 to nucleotideresidue 266042 of the complete genome of M. genitalium (as detailed inAccession No. NC 000908, M. genitalium G-37). Thus, the nucleic acidsequences that are located upstream of the complement of SEQ ID NO: 1 inthe M. genitalium genome have nucleotide numbers 1-265595.

Alternatively, the forward primer may bind to a target site thatoverlaps the complement of SEQ ID NO: 1 and upstream nucleic acidsequences that flank the complement of SEQ ID NO: 1 in the M. genitaliumgenome.

Upstream nucleotides are typically designated using negative numbers. Byway of example, the nucleotide residue 11 residues upstream from thestart of the complement of SEQ ID NO: 1 in the M. genitalium genome(i.e. nucleotide residue 265585 of the complete M. genitalium genome) isdesignated “nucleotide −11”.

The nucleic acid sequence of the 60 nucleotide residues located upstreamfrom SEQ ID NO: 1 in the M. genitalium genome (i.e. nucleotides −60 to−1 of SEQ ID NO: 1) are represented by SEQ ID NO: 6. Thus, nucleotideresidue number 1 of SEQ ID NO: 6 is nucleotide residue number −60 of SEQID NO: 1, and nucleotide residue number 60 of SEQ ID NO: 6 is nucleotideresidue number −1 of SEQ ID NO: 1.

In one aspect, the forward primer binds to a target site located betweennucleotides −80 to +450 of a nucleic acid strand complementary to SEQ IDNO: 1. Within this range of nucleotide residues, the target site for theforward primer is preferably located from nucleotide residue −76 of anucleic acid strand complementary to SEQ ID NO: 1, and may be locatedfrom nucleotide residue −60, −55, −53, −50, −25, −10, 1, 25, 51, 101,151, 201, 251, 301, 351 or 401 of a nucleic acid strand complementary toSEQ ID NO: 1. Preferably, the target site for the forward primer islocated up to nucleotide residue 447 of a nucleic acid strandcomplementary to SEQ ID NO: 1, and may be located up to nucleotideresidue 425, 400, 350, 300, 250, 200, 150, 100, 50, 25, 1, −5, −10, −25,−50 or −55 of a nucleic acid strand complementary to SEQ ID NO: 1.

In one aspect, the forward primer binds to a target site located betweennucleotide residues −25 to 25 of a nucleic acid strand complementary toSEQ ID NO: 1. Preferably, the target site for the forward primer islocated from nucleotide residue −20, −15, −14, −13 or −12 and up tonucleotide residue 22, or 19 of a nucleic acid strand complementary toSEQ ID NO: 1.

In one aspect, the target site for the forward primer is defined bynucleotide residues −11 to 18 of a nucleic acid strand complementary toSEQ ID NO: 1. This target region consists of nucleotide residues 50-60of a nucleic acid strand complementary to SEQ ID NO: 6, followed bynucleotide residues 1-18 of a nucleic acid strand complementary to SEQID NO: 1.

For the avoidance of any doubt, the above numbering system applied tothe nucleic acid residues of the complementary strand of SEQ ID NO: 1(and upstream sequences, SEQ ID NO: 6) is based on the numbering of thenucleic acids of SEQ ID NO: 1 (and upstream sequences, SEQ ID NO: 6) towhich they are complementary.

The reverse primer binds to a target site within the M. genitaliumnucleic acid. This target site may be located within SEQ ID NO: 1—i.e.the target site may be located between nucleotide 1 and nucleotide 447of SEQ ID NO: 1.

Reference to “a target site between (for example) nucleotide residues 1and 447 of SEQ ID NO: 1” does not imply that the target site extendsbetween or is defined by the recited residues. Thus, the target site forthe reverse primer may be 447 nucleotides long, or may be shorter, evenconsiderably shorter than 447 nucleotides in length (e.g. up to 200,100, 75, 60, 50, 40, 30, 25, 20, nucleotides long), so long as it islocated somewhere within the recited range of nucleotide residues.

In one aspect, the target site for the reverse primer is about 50-55nucleotides long. In one aspect, the target site for the reverse primeris about 25-30 nucleotides long, preferably about 27 nucleotides long.

Alternatively, the target site for the reverse primer within the M.genitalium nucleic acid may be located within downstream nucleic acidsequences that flank SEQ ID NO: 1 in the M. genitalium genome.

SEQ ID NO: 1 is located from nucleotide residue 265596 to nucleotideresidue 266042 of the complete genome of M. genitalium (as detailed inAccession No. NC 000908, M. genitalium G-37). Thus, the nucleic acidsequences that are located downstream of SEQ ID NO: 1 in the M.genitalium genome have nucleotide numbers 266043 to 580074.

Alternatively, the reverse primer may bind to a target site thatoverlaps SEQ ID NO: 1 and downstream nucleic acid sequences that flankSEQ ID NO: 1 in the M. genitalium genome.

Downstream nucleotides are designated with positive numbers, continuingfrom the number of the last nucleotide of SEQ ID NO: 1 (residue 447). Byway of example, the nucleotide residue located 20 residues downstreamfrom the end of SEQ ID NO: 1 in the M. genitalium genome (i.e.nucleotide residue 266063 of the complete M. genitalium genome) isdesignated “nucleotide +467” (i.e. 447+20=467).

The nucleic acid sequence of the 53 nucleotide residues locateddownstream from SEQ ID NO: 1 in the M. genitalium genome (i.e.nucleotides +448 to +500 of SEQ ID NO: 1) are represented by SEQ ID NO:7. Thus, nucleotide residue number 1 of SEQ ID NO: 7 is nucleotideresidue number +448 of SEQ ID NO: 1, and nucleotide residue number 53 ofSEQ ID NO: 7 is nucleotide residue number +500 of SEQ ID NO: 1.

In one aspect of the invention, the reverse primer binds to a targetsite between nucleotide residues −5 to +525 of SEQ ID NO: 1. Within thisrange of nucleotide residues, the target site for the reverse primer ispreferably located from nucleotide residue −3 of SEQ ID NO: 1, and maybe located from nucleotide residue 1, 10, 25, 48, 98, 148, 198, 248,298, 348, 398, +448 or +503 of SEQ ID NO: 1. Preferably, within thisrange of nucleotide residues, the target site for the reverse primer islocated up to nucleotide residue +502 of SEQ ID NO: 1, and may belocated up to nucleotide residue 447, 397, 347, 297, 247, 197, 147, 97,47 or 25 of SEQ ID NO: 1.

In one aspect, the reverse primer binds a target site between nucleotideresidues 200 to 250 of SEQ ID NO: 1. Within the range of nucleotideresidues, the target site for the reverse primer is preferably locatedfrom nucleotide residue 205, 210, 211, 212 or 213 of SEQ ID NO: 1 and upto nucleotide residue 245, 244, 243, 242 or 241 of SEQ ID NO: 1. In oneaspect, the reverse primer binds to a target region defined by residues214 to 240 of SEQ ID NO: 1.

The amplified nucleic acid sequence (amplicon) is preferably at least 10nucleotides long, more preferably at least 20 nucleotides long, morepreferably at least 40 nucleotides long, and may be at least 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375 or 400nucleotides long. The amplicon is preferably up to 440 nucleotides long,preferably up to 425 nucleotides long, more preferably up to 400nucleotides long, and may be up to 375, 350, 325, 300, 275, 250, 225,200, 175, 150, 125, 100, 75, 50, 40 or 20 nucleotides long. In oneaspect, the amplicon is about 225-275 nucleotides long, preferably about251 nucleotides long.

Particularly good results have been obtained using a forward primerselected from SEQ ID NOs: 4, 8 or 31, as shown in the table below, whichbind to a target site defined by nucleotide residues −11 to 18 of anucleic acid strand complementary to SEQ ID NO: 1.

SEQ ID NO: SEQUENCE 4 5′ GAC AGT TCA TTA TGC GCA CCA GTT ACT TG 3′ 85′ CAT AGT TCA TTA TGC GCA CCA GTT ACT TG 3′ 31 5′ CAT AGT TCA TTA TGCACA CCA GTT ACT TG 3′

Particularly good results have been obtained using a forward primerselected from SEQ ID NOs: 9-19, as shown in the table below (see alsoExample 4).

9 5′ CCA CTT AAC TTT ATT ACC CGT CC 3′ 10 5′ TGT TTT CAA AAG TAA TTT GCCACC GAA ACT AAG TAA GGA TGA CAT AGT TCA TT 3′ 11 5′ ATG CGC ACC AGT TACTTG AAA AAA ATA CCC ATA ATG AAT AGT GAT AGT GA 3′ 12 5′ TCT AAA ACT CCAAAA GGT GTG GAT CGA GCG GCA TGT TGA TCA AGA TGA AC 3′ 13 5′ TTA GTT TAACAA CTA CTG CAG TTG AAC TTA AAA AGA GTG ATG AAC AAA AA 3′ 14 5′ CCT GTTGCC ATT AAA AGT AGT GAC TTT ATT GGT CAT GAA GAG TTA ATC TC 3′ 15 5′ TGTTCC AGT TTT ACT AAT CCC AAC CCC TGT TGT TAA AGA GAT TGA TCA AC 3′ 165′ CAG CAG TTA TTC CTC CAG TTA AAG CAA AAC CAA AAG CAA CTA AAA AGA AA 3′17 5′ ACT CCT GTT AAA TCA AAA CCA ACT AGT AAA TCA ACT AAA CAA ACA AAA CC3′ 18 5′ TAA ACA ATC CAA GCC CAA ATC AAA ACA AGT TCA ACA AAC CAA AGC TAAAC 3′ 19 5′ CAA CCC AAA TTC AAA CAA AAA AAA GCA ATA AAA AAA CCA GAT CTTAAT CT 3′

Particularly good results have been obtained using a reverse primer ofSEQ ID NO: 5, as shown in the table below, which binds to a target sitedefined by nucleotide residues 214 to 240 of SEQ ID NO: 1.

SEQ ID NO: SEQUENCE 5 5′ CTC TTT AAC AAC AGG GGT TGG GAT TAG 3′

Particularly good results have been obtained using a reverse primerselected from SEQ ID NOs: 20-30, as shown in the table below (see alsoExample 4).

20 5′ GAT TAA CCC CAG GTA GTT CTT CC 3′ 21 5′ TAT TCC TTT CCA GTT TTTAGT TAA AAC TAC TGT TGT TAA CAC TAA AAA AAC CAG A3′ 22 5′ TTA AGA TCTGGT TTT TTT ATT GCT TTT TTT TGT TTG AAT TTG GGT TGG TT 3′ 23 5′ TAG CTTTGG TTT GTT GAA CTT GTT TTG ATT TGG GCT TGG ATT GTT TAG GT 3′ 24 5′ TTTGTT TGT TTA GTT GAT TTA CTA GTT GGT TTT GAT TTA ACA GGA GTT TT 3′ 255′ CTT TTT AGT TGC TTT TGG TTT TGC TTT AAC TGG AGG AAT AAC TGC TGG TT 3′26 5′ GAT CAA TCT CTT TAA CAA CAG GGG TTG GGA TTA GTA AAA CTG GAA CAG AG3′ 27 5′ ATT AAC TCT TGA TGA CCA ATA AAG TCA CTA CTT TTA ATG GCA ACA GGTTT 3′ 28 5′ TTG TTC ATC ACT CTT TTT AAG TTC AAC TGC AGT AGT TGT TAA ACTAAG TT 3′ 29 5′ CAT CTT GAT CAA CAT GCC GCT CGA TCC ACA CCT TTT GGA GTTTTA GAT CA 3′ 30 5′ CTA TCA CTA TTC ATT ATG GGT ATT TTT TTC AAG TAA CTGGTG CGC ATA AT 3′

It will, however, be appreciated that variants may be employed, whichdiffer from the above-mentioned primer sequences by one or morenucleotides. In this regard, conservative substitutions are preferred.

Thus, in one aspect, the forward primer comprises a nucleic acidsequence having at least 60%, preferably at least 65%, preferably atleast 70%, preferably at least 75%, preferably at least 80%, preferablyat least 85%, preferably at least 90%, preferably at least 95%, morepreferably at least 97%, most preferably 100% sequence identity to asequence selected from SEQ ID NOs: 4, 8-19 or 31.

Thus, in one aspect, the reverse primer comprises a nucleic acidsequence having at least 60%, preferably at least 65%, preferably atleast 70%, preferably at least 75%, preferably at least 80%, preferablyat least 85%, preferably at least 90%, preferably at least 95%, morepreferably at least 97%, most preferably 100% sequence identity to asequence selected from SEQ ID NO: 5 or SEQ ID NOs: 20-30.

Preferably, the forward primer consists of a nucleic acid sequencehaving at least 60%, preferably at least 65%, preferably at least 70%,preferably at least 75%, preferably at least 80%, preferably at least85%, preferably at least 90%, preferably at least 95%, more preferablyat least 97%, most preferably 100% sequence identity to a sequenceselected from SEQ ID NO: 4, 8-19 or 31.

Preferably, the reverse primer consists of a nucleic acid sequencehaving at least 60%, preferably at least 65%, preferably at least 70%,preferably at least 75%, preferably at least 80%, preferably at least85%, preferably at least 90%, preferably at least 95%, more preferablyat least 97%, most preferably 100% sequence identity to a sequenceselected from SEQ. ID NOs: 5 or 20-30.

Fragments of the above-mentioned primer sequences may also be employed.

Preferably, a fragment of forward primers SEQ ID NOs: 4, 8 and 31comprises at least 15 consecutive nucleotides of said primer sequences,more preferably at least 20, 21, 22, 23, 24, 25, 26 or 27 consecutivenucleotides of said primer sequences, and may comprise up to 28consecutive nucleotides of said primer sequences.

Preferably, a fragment of reverse primer SEQ ID NO: 5 comprises at least15 consecutive nucleotides of said primer sequence, more preferably atleast 20, 21, 22, 23, 24 or 25 consecutive nucleotides of said primersequence, and may comprise up to 26 consecutive nucleotides of saidprimer sequence.

Preferably, a fragment of primer SEQ ID NOs: 9 and 20 comprises at least10 consecutive nucleotides of said primer sequences, more preferably atleast 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of saidprimer sequences, and may comprise up to 22 consecutive nucleotides ofsaid primer sequences.

Preferably, a fragment of primer SEQ ID NOs: 10-30 comprises at least 30consecutive nucleotides of said primer sequences, more preferably atleast 35, 40, 41, 42, 43, 44, 45, 46, 47 or 48 consecutive nucleotidesof said primer sequences, and may comprise up to 49 consecutivenucleotides of said primer sequences.

In the method of the present invention, any forward primer selected fromSEQ ID NOs: 4, 8-19 or 31 (or fragments thereof, or variants thereofhaving a % identity thereto as discussed above) may be used incombination with any reverse primer selected from SEQ ID NOs: 5 or 20-30(or fragments thereof, or variants thereof having a % identity theretoas discussed above).

In one aspect, the method may employ forward primer SEQ ID NO: 4, 8 or31 and reverse primer SEQ ID NO: 5 (or fragments thereof, or variantsthereof having % identity thereto as discussed above).

In one aspect, the method may employ forward primer SEQ ID NO: 9 andreverse primer SEQ ID NO: 20 (or fragments thereof, or variants thereofhaving % identity thereto as discussed above).

It is an option for at least one of the primers to comprise a minorgroove binder (MGB) component.

The detection step may be carried out by any known means. In one aspect,the amplified nucleic acid sequence is detected by a method comprisinggel electrophoresis.

Alternatively, or in addition, the step of detecting the amplifiednucleic acid sequence may comprise contacting said amplified nucleicacid sequence with a probe, wherein the probe binds to a target sitewithin said amplified nucleic acid sequence, or the complement thereof,and detecting binding of said probe to said amplified nucleic acidsequence.

Suitable probes for use in the methods of the present invention areligands that bind specifically to M. genitalium nucleic acid. Suchligands may be oligonucleotide ligands or protein ligands, for example,antibodies. However, it is preferred that the probes are oligonucleotideprobes.

For the avoidance of doubt, in the context of the present invention, thedefinition of an oligonucleotide probe does not include the full lengthMg219 gene (or complement thereof).

Probes are designed to bind to the target gene sequence (i.e. within theamplicon, or within SEQ ID NO: 1) based on a selection of desiredparameters, using conventional software. It is preferred that thebinding conditions are such that a high level of specificity isprovided—i.e. binding occurs under “stringent conditions”. In general,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(n), is the temperature (under definedionic strength and pH) at which 50% of the target sequence binds to aperfectly matched probe. In this regard, the T_(m) of probes of thepresent invention, at a salt concentration of about 0.02 M or less at pH7, is preferably above 60° C., more preferably about 70° C. Premixedbinding solutions are available (e.g. EXPRESSHYB Hybridisation Solutionfrom CLONTECH Laboratories, Inc.), and binding can be performedaccording to the manufacturer's instructions. Alternatively, a personskilled in the art can devise variations of these binding conditions.

Following binding, washing under stringent (preferably highly stringent)conditions removes unbound nucleic acid molecules. Typical stringentwashing conditions include washing in a solution of 0.5-2×SSC with 0.1%SDS at 55-65° C. Typical highly stringent washing conditions includewashing in a solution of 0.1-0.2×SSC with 0.1% SDS at 55-65° C. Askilled person can readily devise equivalent conditions for example, bysubstituting SSPE for the SSC in the wash solution.

It is preferable to screen the probes to minimise self-complementarityand dimer formation (probe-probe binding). Preferred probes of thepresent invention are selected so as to have minimal homology with humanDNA. The selection process may involve comparing a candidate probesequence with human DNA and rejecting the probe if the homology isgreater than 50%. The aim of this selection process is to reduceannealing of probe to contaminating human DNA sequences and hence allowimproved specificity of the assay.

In one embodiment, conjugation of a minor groove binder (MGB) to theprobe stabilises nucleic acid duplexes, causing a desirable increase inoligonucleotide Tm (the temperature at which the duplex separates). Thisincrease in Tm enables considerably shorter probes to be used than wouldotherwise be possible.

In one aspect, the oligonucleotide probe is at least 5 nucleotides long,more preferably at least 10 nucleotides long, more preferably at least12, 13, 14, 15 or 16 nucleotides long. Preferably, the probe is up to 50nucleotides long, more preferably up to 30 nucleotides long, and morepreferably up to 20, 19 or 18 nucleotides long. It is an advantage touse shorter probes, as this enables faster annealing to target M.genitalium nucleic acid.

The complement of a nucleic acid sequence binds via complementarybase-pairing to the nucleic acid sequence. In the present invention, a“complementary strand” means the anti-sense nucleic acid strand, whichbinds via complementary base-pairing to a sense strand.

Thus, in one aspect, the probe binds to a target sequence within thecoding (sense) strand of the target M. genitalium nucleic acid (i.e.within SEQ ID NO: 1). In an alternative aspect, the probe binds to atarget sequence within the complementary, non-coding (anti-sense) strandof the target M. genitalium nucleic acid (i.e. within the complement ofSEQ ID NO: 1).

The target site to which the probe binds may be located anywhere withinSEQ ID NO: 1, or within the complement of SEQ ID NO: 1. If the nucleicacid in the sample has previously been amplified, the probe binds to atarget site within said amplified nucleic acid sequence, or thecomplement thereof.

Thus, in one aspect, the probe binds to a target site located anywherebetween residues 1 and 447 of SEQ ID NO: 1, or the complement thereof.In this regard, reference to “a target site between (for example)residues 1 and 447 of SEQ ID NO: 1 or the complement thereof” does notimply that the target site extends between or is defined by the recitedresidues. Thus, the target site may be 447 nucleotides long, or may beshorter, even considerably shorter than 447 nucleotides in length (e.g.up to 200, 100, 75, 60, 50, 40, 30, 25, 20, 15 or 10 nucleotides long).

Thus, in one aspect, the probe binds to a target site between nucleicacid residues 1-447 of SEQ ID NO: 1 or the complement thereof. Withinthis range of nucleotide residues, the target site for the probe ispreferably located from nucleotide residue 10 of SEQ ID NO: 1 or thecomplement thereof, more preferably from nucleotide residue 15 of SEQ IDNO: 1 or the complement thereof, and may be located from nucleotideresidue 25, 50, 75, 100, 150, 200, 250, 300, 350 or 400 of SEQ ID NO: 1or the complement thereof. Within this range of nucleotide residues, thetarget site for the probe is preferably located up to nucleotide residue440, more preferably up to nucleotide residue 420 of SEQ ID NO: 1 or thecomplement thereof, and may be located up to residue 400, 350, 300, 250,200, 150, 100 or 50 of SEQ ID NO: 1 or the complement thereof.

In one aspect, the probe binds to a target region located betweennucleotide residues 50-100 of the nucleic acid strand complementary toSEQ ID NO: 1. Preferably, the target site for the probe is located fromnucleotide residue 55, 60, 63, 64 or 65 and up to nucleotide residue 90,85, 84 or 83 of the nucleic acid strand complementary to SEQ ID NO: 1.Most preferably, the target site for the probe is defined by residues66-82 of the nucleic acid strand complementary to SEQ ID NO: 1.

For the avoidance of any doubt, the above numbering system applied tothe nucleic acid residues of the complementary strand of SEQ ID NO: 1 isbased on the numbering of the nucleic acids of SEQ ID NO: 1 to whichthey are complementary.

Good results have been obtained using a probe consisting of the nucleicacid sequence SEQ ID NO: 3, as shown in the table below.

SEQ ID NO: SEQUENCE 3 5′-GGT GTG GAT CGA GCG GC-3′

It will, however, be appreciated that variants may be employed, whichdiffer from the above-mentioned probe sequence by one or morenucleotides. In this regard, conservative substitutions are preferred.

Thus, in one aspect, the probe comprises a nucleic acid sequence havingat least 60%, preferably at least 65%, preferably at least 70%,preferably at least 75%, preferably at least 80%, preferably at least85%, preferably at least 90%, preferably at least 95%, most preferably100% sequence identity to SEQ ID NO: 3.

In one aspect, the probe consists of a nucleic acid sequence having atleast 60%, preferably at least 65%, preferably at least 70%, preferablyat least 75%, preferably at least 80%, preferably at least 85%,preferably at least 90%, preferably at least 95%, most preferably 100%sequence identity to SEQ ID NO: 3.

An alternative means for defining variant probe sequences is by definingthe number of nucleotides that differ between the variant sequence andthe probe sequence of the present invention. In this regard, the presentinvention embraces probe sequences that differ from SEQ ID NO: 3 by nomore than 6 nucleotides, preferably by no more than 5 nucleotides,preferably by no more than 4 nucleotides, more preferably by no morethan 3 nucleotides, yet more preferably by no more than 2 nucleotides,and most preferably by no more than 1 nucleotide.

A fragment of the above-mentioned probe sequence may also be employed,wherein the fragment comprises at least 10, preferably at least 11, 12,13, 14, or 16 consecutive nucleotides of SEQ ID NO: 3.

In one aspect, the probe may be immobilised onto a solid support orplatform. The support may be a rigid solid support made from, forexample, glass or plastic, or else the support may be a nylon ornitrocellulose membrane, or other membrane. 3D matrices are suitablesupports for use with the present invention—e.g. polyacrylamide or PEGgels. In one embodiment, the solid support may be in the form of beads,which may be sorted by size or fluorophores.

The probes may be immobilised to the solid support by a variety ofmeans. By way of example, probes may be immobilised onto a nylonmembrane by UV cross-linking. Biotin-labelled probes may be bound tostreptavidin-coated substrates, and probes prepared with amino linkersmay be immobilised onto silanised surfaces.

Another means of immobilising probe is via a poly-T tail, preferably atthe 3′ end. The poly-T tail consists of a run of from 1 to 100 thymineresidues added to the probe at the 3′ end with a terminal transferase.Preferably, from 1 to 20 thymine residues are added. The poly-T tail isthen baked or UV cross-linked onto the solid substrate. Addition of apoly-T tail appears to have two functions. First, the poly-T tailincreases the amount of probe that is immobilised onto the solidsupport. Second, the poly-T tail conforms the probe in such a way as toimprove the efficiency of hybridisation.

It is an option for the probe to comprise a minor groove binder (MGB)component.

In one aspect, binding of probe to M. genitalium nucleic acid provides adetectable signal, which may be detected by known means. A detectablesignal may be, for example, a radioactive signal or a fluorescentsignal, such as a change in fluorescence.

In one embodiment, the probe is labelled and the assay comprisesdetecting the label and correlating presence of label with presence ofM. genitalium nucleic acid. The label may be a radiolabel but ispreferably non-radioactive, such as a fluorescent label. By way ofexample, the label may be digoxygenin or fluorescein-isothiocyanate(FITC).

The label may be detected directly, such as by exposure to photographicor X-ray film, or indirectly, for example, in a two-phase system. Anexample of indirect label detection is binding of an antibody to thelabel. In another aspect, the probe is labelled with biotin and isdetected using streptavidin bound to a detectable molecule or to anenzyme, which generates a detectable signal.

In one aspect, prior to detecting binding of the probe to its targetsequence, the method comprises amplifying M. genitalium nucleic acid. Itmay be desirable to amplify the target human papillomavirus nucleic acidif the sample is small and/or comprises a heterogeneous collection ofDNA sequences.

Most preferably, the amplification step is carried out prior tocontacting the nucleic acid with the probe.

Amplification of M. genitalium nucleic acid is preferably carried out asdescribed above. In this regard, the amplification is preferably carriedout by contacting the sample with forward and reverse oligonucleotideprimers as described above. Said primers bind to target sites in the M.genitalium nucleic acid, under conditions suitable to promoteamplification of a nucleic acid sequence comprising at least the targetsequence to which the probe binds within SEQ ID NO: 1.

The present invention enables quantitative estimates of pathogen load tobe determined. Determining pathogen load has many useful applications,such as for clinical guidance and for determining therapy.

Thus, in one aspect, the present invention provides a method ofquantitating M. genitalium pathogen load in a sample of interest,comprising: (a) carrying out a detection method according to the presentinvention on said sample of interest; and (b) carrying out said methodon a test sample of predetermined known M. genitalium pathogen load; and(c) comparing the signal detected from the sample of interest with thesignal detected from the test sample; and thereby quantitating M.genitalium pathogen load in the sample of interest.

In another aspect, the method of the present invention is useful fordetermining efficacy of a course of treatment over a period of time, forexample a course of drug therapy.

Thus, in one aspect, the present invention provides an in vitro methodof determining drug efficacy over the course of a period of drugtherapy, comprising: (a) carrying out the present method on a firstsample obtained at a first time point within or prior to the period ofdrug therapy; (b) carrying out the present method on a sample at one ormore later time points within the period of drug therapy; and (c)comparing the signal detected from the first sample with the signaldetected from the one or more later samples; and thereby determiningdrug efficacy over the course of the period of drug therapy.

In one aspect, the method of the present invention is useful fordetecting and/or monitoring the development of resistance to a drug(e.g. an anti-microbial such as an antibiotic for M. genitalium) over aperiod of time.

Thus, in one aspect, the present invention provides an in vitro methodof detecting and/or monitoring the development of resistance of M.genitalium to a drug, comprising (a) carrying out the present method ona first sample obtained at a first time point within or prior toexposure of the M. genitalium to the drug; (b) carrying out the presentmethod on a sample at one or more later time points following exposureof the M. genitalium to the drug; and (c) comparing the signal detectedfrom the first sample with the signal detected from the one or morelater samples; wherein the absence of a reduction in the signal, or anincrease in the signal, indicates that the M. genitalium have developedresistance to the drug.

In one aspect, the method of the present invention is useful forscreening human semen and/or human eggs prior to artificialinsemination, to confirm the presence or absence of M. genitalium. Inthis regard, confirmation of the absence of M. genitalium infection maybe required prior to IVF treatment. Hence, the method of the presentinvention advantageously reduces (and preferably substantiallyeliminates) the risk of transmitting M. genitalium to a patientundergoing in vitro fertilisation (IVF) treatment.

Thus, in one aspect, the present invention provides an in vitro methodof screening a sample comprising human semen and/or eggs for M.genitalium, comprising carrying out the present detection method;wherein the absence of a signal indicates that the sample is free of M.genitalium.

The detection method of the present invention may also be useful forscreening people undergoing IVF treatment, and surrogate mothers, for M.genitalium infection.

The detection method of the present invention may also be useful foroccupational screening for M. genitalium infection. The detection methodof the present invention may also be useful for forensic testing e.g. inrap, sexual assault or child abuse cases.

The invention also provides reagents for use in the above-describedmethods of the present invention.

Hence, the present invention provides a forward primer as describedabove for use in accordance with the invention. The present inventionalso provides a reverse primer as described above for use in accordancewith the invention. The present invention also provides a pair offorward and reverse oligonucleotide primers selected from the primersdescribed above in accordance with the invention.

The present invention provides a probe as described above for use inaccordance with the present invention.

Also provided by the present invention is a kit for detecting M.genitalium nucleic acid, comprising a forward primer as described above.Optionally, the kit includes a reverse primer as described above.Optionally, the kit includes a probe as described above.

The present invention is discussed in more detail by means of theExamples described below, and by the Figures.

FIG. 1 illustrates the relationship between the Mg219 gene (SEQ ID NO:1), probe SEQ ID NO: 3, forward primer SEQ ID NO: 4, 8 or 31, andreverse primer SEQ ID NO: 5.

In more detail, FIG. 1 illustrates the 447 nucleotide sequence SEQ IDNO: 1 with 3 superimposed shaded regions representing the target/bindingsites for the probe SEQ ID NO: 3, the forward primer SEQ ID NO: 4, 8 or31, and the reverse primer SEQ ID NO: 5. In this regard, although theprobe SEQ ID NO: 3 and the forward primer SEQ ID NO: 4, 8 or 31 bind tothe complement of SEQ ID NO: 1, for the sake of clarity, the targetsites of the probe and forward primer are illustrated with reference toSEQ ID NO: 1.

Thus, the first shaded region represents the target site to which theforward primer SEQ ID NO: 4, 8 or 31 binds, i.e. nucleotide residues −11to 18 of the complement of SEQ ID NO: 1. The second shaded regionrepresents the target site for the probe SEQ ID NO: 3, i.e. nucleotideresidues 66-82 within the complement of SEQ ID NO: 1. The third shadedregion represents the target site to which the reverse primer SEQ ID NO:5 binds, i.e. nucleotide residues 214 to 240 of SEQ ID NO: 1.

Thus, FIG. 1 illustrates that using a forward primer of SEQ ID NO: 4, 8or 31, and a reverse primer of SEQ ID NO: 5, the resulting amplicon is251 nucleotides long (spanning nucleotide residue −11 to nucleotideresidue 240).

FIG. 2 illustrates the relationship between the Mg219 gene (SEQ ID NO:1), forward primers SEQ ID NOs: 9-19 and reverse primers SEQ ID NOs:20-30.

In more detail, FIG. 2 illustrates the 447 nucleotide sequence of Mg219i.e. SEQ ID NO: 1 (central shaded bar, annotated every 100 nucleotides)plus 100 upstream nucleotides and over 500 downstream nucleotides.Shaded arrows 9 and 20 represent the target/binding sites for theupstream and downstream sequencing primers SEQ ID NOs: 9 and 20,respectively. Shaded arrows 10-19 represent the target/binding sites forthe forward primers SEQ ID NOs: 10-19. Shaded arrows 21-30 represent thetarget/binding sites for the reverse primers SEQ ID NOs: 21-30. In thisregard, although the forward primers bind to the complement of SEQ IDNO: 1, for the sake of clarity, the target sites of the forward primerare illustrated with reference to SEQ ID NO: 1.

Thus, it is evident from FIG. 2 that primer SEQ ID NOs: 9 and 10 bind totarget sequences located entirely within nucleotide sequences that areupstream of the complement of the Mg219 nucleic acid sequence. Likewise,primer sequences 20 and 21 bind to target sequences located entirelywithin nucleotide sequences that are downstream of the Mg219 nucleicacid sequence. In contrast, primer SEQ ID NOs: 11-18 bind to targetsequences located entirely within the complement of the Mg219 nucleicacid sequence, and primer SEQ ID NOs: 22-29 bind to target sequenceslocated entirely within the Mg219 nucleic acid sequence. However, thetarget site for primer SEQ ID NO: 19 overlaps the complement of theMg219 nucleic acid sequence and (3) nucleotides located downstream ofthe complement of the Mg219 nucleic acid sequence. Likewise, the targetsite for primer SEQ ID NO: 30 overlaps the Mg219 nucleic acid sequenceand (3) nucleotides located upstream of the complement of the Mg219nucleic acid sequence.

FIG. 3 illustrates the results of gradient PCR to determine optimalannealing temperature using primer sequences SEQ ID NO: 9 and SEQ ID NO:20. The temperatures tested are as follows: A=57.0° C., B=57.3° C.,C=58.0° C., D=58.8° C., E=60.1° C., F=61.8° C., G=63.6° C., H=65.5° C.,I=66.4° C., J=67.2° C., K=control.

FIG. 4 illustrates detection of the entire Mg219 gene using a span ofoligonucleotide primers (forward primer SEQ ID NO: 9 with reverseprimers selected from SEQ ID NOs: 20-30) across the full gene sequence.The primer pairs employed are as follows:

-   -   A=SEQ ID NO: 9 plus SEQ ID NO: 30    -   B=SEQ ID NO: 9 plus SEQ ID NO: 29    -   C=SEQ ID NO: 9 plus SEQ ID NO: 28    -   D=SEQ ID NO: 9 plus SEQ ID NO: 27    -   E=SEQ ID NO: 9 plus SEQ ID NO: 26    -   F=SEQ ID NO: 9 plus SEQ ID NO: 25    -   G=SEQ ID NO: 9 plus SEQ ID NO: 24    -   H=SEQ ID NO: 9 plus SEQ ID NO: 23    -   I=SEQ ID NO: 9 plus SEQ ID NO: 22    -   J=SEQ ID NO: 9 plus SEQ ID NO: 21    -   K=SEQ ID NO: 9 plus SEQ ID NO: 20

FIG. 5 illustrates detection of the entire Mg219 gene using a span ofoligonucleotide primers (reverse primer SEQ ID NO: 20 with forwardprimers selected from SEQ ID NOs: 9-19) across the full gene sequence.The primer pairs employed are as follows:

-   -   A=SEQ ID NO: 20 plus SEQ ID NO: 10    -   B=SEQ ID NO: 20 plus SEQ ID NO: 11    -   C=SEQ ID NO: 20 plus SEQ ID NO: 12    -   D=SEQ ID NO: 20 plus SEQ ID NO: 13    -   E=SEQ ID NO: 20 plus SEQ ID NO: 14    -   F=SEQ ID NO: 20 plus SEQ ID NO: 15    -   G=SEQ ID NO: 20 plus SEQ ID NO: 16    -   H=SEQ ID NO: 20 plus SEQ ID NO: 17    -   I=SEQ ID NO: 20 plus SEQ ID NO: 18    -   J=SEQ ID NO: 20 plus SEQ ID NO: 19    -   K=SEQ ID NO: 20 plus SEQ ID NO: 9    -   L=Negative control

FIG. 6 illustrates amplification of Mg219 from 12 cultured M. genitaliumstrains (identified as 1-12, see Example 5 for key), using the primerpair SEQ ID NO: 9 and SEQ ID NO: 20.

FIG. 7 presents an alignment of the Mg219 gene (plus upstream anddownstream nucleotides) from M. genitalium strain G37 plus 16 culturedisolates, illustrating the binding sites for the forward and reversesequencing primers (SEQ ID NOs: 9 and 20), the forward assay primer (SEQID NO: 4, 8 or 31), the reverse assay primer (SEQ ID NO: 5) and theprobe (SEQ ID NO: 3). Each of the aligned sequences has been allocated aSEQ ID NO (see Example 5, below).

FIG. 8 is a ClustalW (v1.4) multiple sequence alignment matrix,illustrating the sequence identity level between the Mg219 sequences(plus up- and down-stream nucleotides) illustrated in FIG. 7. AlignmentScore=477772, Gaps Inserted=30, Conserved Identities=549. PairwiseAlignment Mode: Fast. Pairwise Alignment Parameters: ktup=1, GapPenalty=3, Top Diagonals=5 Window Size=5. Multiple Alignment Parameters:Open Gap Penalty=10.0, Extend Gap Penalty=0.1, Delay Divergent=40%Transitions: Weighted.

EXAMPLES Example 1 Standard Operating Procedure for Detecting M.genitalium Mg219 Gene

This Example details the procedures for the detection of M. genitaliumMg219 gene DNA from clinical material or cultures by Polymerase ChainReaction (PCR) using fluorescent probes on the Corbett Rotor-genereal-time PCR machine.

Gloves and safety glasses were worn throughout, and all extractionprocedures were carried out in a Class 1 Safety cabinet. Lowerrespiratory tract specimens such as sputa and lung biopsies were handledin a CL3 facility while other samples were processed in a Class 1 SafetyCabinet.

Preparing and Running the PCR

Immediately prior to loading tubes into the Rotor-gene PCR machine, thetemplate sample was added to the mixture in a designated PCR cabinet inthe PCR clean room. PCR tubes containing mixtures prepared for cyclingwere capped before leaving the cabinet and conveyed to the Rotor-gene.

Materials

Sterile screw cap 1.5 mL microfuge plastic tubes

Sputasol™. (Oxoid) freshly diluted in water (Sigma)

QIA-Amp DNA Mini Kit. (Qiagen cat no 51306)

Gilson pipettes: P1000, P200, P20, P10 (or equivalent)

Pre-sterile tips with filters for above (Rainin)

Corbett Rotagene Realtime PCR machine

0.5 mL PCR tubes (flat top)

Tris-EDTA (TE) buffer 100× concentrate (Sigma cat no T9285)

AmpliTaq Gold@ DNA Polymerase, with GeneAMP (Applied Biosystems cat no4311820)

AmpErase® Uracil N-glycosylase (UNG) (Applied Biosystems cat noN8080096)

Heating block 56±2° C.

Primers and probes: Adjusted to give 100 μM stock solutions, aliquoted(504) and stored at −30° C. or below.

M. genitalium NCTC 10195^(T) (Positive control) cloned stocks of DNAtemplate (pGEMT-EASY genit219 POS 1, 2, 3, 4 or 5 in E. coli TOP10^(F1))

GeneQuant.II (Pharmacia Biotech)

Nuclease-free water (Promega cat no P119C)

Herring sperm DNA

dNTP Blend, 12.5 mM with dUTP (Applied Biosystems cat no N8080270)

Procedures

Samples received for PCR may be tested by culture for Mycoplasmas andUreaplasmas (culture samples prior to commencing DNA extraction).

DNA was extracted using a conventional DNA extraction protocol(depending on the circumstances e.g. nature and volume of sample,urgency of request etc. different DNA extraction protocols may be used).

DNA from urine was extracted via centrifugation at 20,000 g for 15 mins,and then the pellet was re-suspended in 0.4 mL PBS and heated at 100° C.for 5 mins (alternatively, the magnapure compact may be used directly on0.5 mL heat inactivated urine (95° C.±10° C.) withoutpre-centrifugation). Swabs were extracted by agitating in 0.4 mL PBS andfollowing the same protocol as above.

PBS or water was extracted in tandem with the clinical sample andincluded in all further tests to ensure cross contamination did notoccur.

A 1:10 dilution of the extracted DNA was prepared in nuclease freewater. For unusual and highly cellular samples such as tissue a 1:10 and1:100 dilution was prepared.

Preparation of Cloned M. genitalium Standard for Regression Calculation

Because of the problems associated with growing sufficient Mycoplasma togenerate stocks of standard DNA, a cloned template DNA sequence wasprepared.

The concentration and purity of the plasmid solution in TE buffer wasestimated using GeneQuant (against TE blank).

The plasmid solution was adjusted to known concentration (100 ng/mL) inHerring sperm DNA at 10 ng/μL (in TE buffer pH 8.0), aliquoted (25 μL),and stored at −20±10° C.

Immediately prior to use, serial dilutions were made (in PCR qualitywater) to cover the expected dynamic range of the clinical specimens anda water blank. 5 ng, 0.5 ng, 0.05 ng and 0.05 ng serial dilutions wereused in each run.

Preparation of Primers and Probes

The primers used in this assay yield a positive product of 251 by for M.genitalium and the probe binds from by 66 to 82 inside the Mg219 gene.

Forward Primer=SEQ ID NO: 4, 8 or 31; Reverse Primer=SEQ ID NO: 5.

Primer aliquots were re-suspended in TE to give 100 pMμL⁻¹, aliquoted,assigned batch numbers and stored at −20±10° C. or below.

Probes (SEQ ID NO: 3, [at the 3 nmol scale]) were re-suspended in TE togive 100 pMμL⁻¹, aliquoted, assigned batch numbers and stored at −20±10°C.

The probes are light sensitive and should therefore be handled indark/amber microfuge tubes at all times.

The working primer/probe mix contains 2.5 pmolμL⁻¹ (2.5 μM) of eachprimer, giving a final concentration of 0.5 μM each and 1 pmol μL⁻¹ (10μM) of M. genitalium probe, giving a final concentration of 0.2 μM. Tomake the mix, the following components were added:

μL (4 mL total) 3760 H₂0 100 Primer Mg219F (SEQ ID NO: 4, 8 or 31) 100Primer Mg219R (SEQ ID NO: 5) 40 Probe Mg219 (SEQ ID NO: 3)

The primer/probe mix was distributed in 10004 aliquots in black tubes,assigned batch numbers and an expiry date of 12 months and stored at−20±10° C.

Preparation of PCR Reagent Mixture.

A stock mastermix was made for 1000 reactions and aliquoted givingenough mastermix for a full carousel (37 reactions). The stock mastermixwas stored at −20±10° C. and given expiry date of 12 months fromformation. The reagents were mixed gently by pipetting before use.

Mastermix Per sample Master mix Final conc. H₂O 3.55 μL 3.55 mL MgCl₂ 25mM 4 μL 4 mL 5 μM dNTPs 12.5 mM 1 μL 1 mL 0.625 mM Primer/Probe mix 4 μL4 mL (0.2 μM MG219 probe, 0.5 μM primer) Amplitaq gold 10 × buffer 2 μL2 mL x1 538.35 μL were distributed into each tube and frozen at −20 ±10° C. On day of use 1 vial was defrosted and the following were addedto 37 reactions: Amplitaq gold polymerase 0.25 μL 9.25 μL 1.25 unitUracil-DNA glycosylase 0.2 μL 7.4 μL 1 unit Total 15 μL 15 μL wasdistributed into each tube.

All manipulations were performed at 2-8° C. A water blank was includedin every PCR experiment. Water was pipetted after other solutions tocontrol for cross contamination. The clinical sample, dilutions andextracted water control were included, pipetted and sealed before addingpositive controls.

54 of the relevant sample were added and the tubes were closed (giving afinal volume of 20 μL). A standard curve of positive control DNA wasincluded. The PCR machine was loaded, making sure that the tubes wereclosed properly by firmly pressing down the lid and always using thelocking rings provided to secure lids on both 0.1 and 0.2 mL tubes.

NOTE: Calibration measures the fluorescence in the 1^(st) tube for allchannels and adjusts fluorescence respectively to take account ofbackground. Therefore the first tube must contain all probes used in theassay. Baseline fluorescence should be ˜20, for quenched FRET assayswhere a decrease in fluorescence is expected it should be 50-70. Setcalibration to be taken at beginning of first cycle to relevant channel.

Detection of PCR Products Setting up the Rotorgene PCR Machine:

The PCR machine was set up, samples were put in and the programme wasentered/opened. Flat tubes and the required programme channels were set(Cy5—channel 4) and the details of specimens and standards in respectivecarousel positions were entered. Save and ‘start’. The entire carouselwas filled with tubes, even if not included in the analysis. The assaywas run.

Cycling conditions Acquisition 45 cycles Temp Time Transition rate modeDenaturation 95° C.  5 min 20 secs None Quantification Segment 1 95° C.15 sec 20° C./sec None 2 60° C. 60 sec 20° C./sec Single Melting CurveSegment 1 95° C.  0 sec 20° C./sec None 2 45° C.  2 sec 20° C./sec None3 80° C.  0 sec 0.1° C./sec  Cont Cooling The Rotor-gene automaticallycools

Data Analysis

By clicking the ‘analysis’ button and double clicking on the Cy5channel, the software gives the standard curve an efficiency value. Ifthey are unexpectedly high, the assay should be repeated.

Example 2 Validation of the Assay

The purpose of the validation was to determine the following:

-   -   1) Specificity—other Mycoplasma species and bacterial species        from same niche    -   2) Sensitivity—determine LDL    -   3) Known positives and negatives    -   4) Optimal extraction method    -   5) Alternative PCR platforms    -   6) Varying sample types    -   7) Reproducibility    -   8) Comparison to MgPa assay

1) Specificity

NCTC/strain DNA Test Species identifier conc result CommensalsLactobacillus vaginalis 12197 — Neg Prevotella (Bacteroides) 11156 1μgmL⁻¹ Neg bivius Mobiluncus curtisii 11657 — Neg Mobiluncus mulieris11658 1 μgmL⁻¹ Neg Gp B Streptococcus (S. pyogenes) 12067 — Neg Gp AStreptococcus (S. algalactiae) 12906 — Neg Echerishia. coli 9001 1μgmL⁻¹ Neg Staphylococcus 8532 — Neg Pathogens Neisseria gonorrhoeaH060160180 — Neg Clinical sample Chlamydia trachomatis L2, Cell culture— Neg DNA Treponema palidum H05510345 — Neg Clinical sample Treponemapalidum Nichols or — Neg Newmarket cell culture strain Heamophilusducreyi PCR control — Neg HSV1 PCR control — Neg Mollicute species A.laidlawii 1 μgmL⁻¹ Neg M. hominis 1 μgmL⁻¹ Neg M. pneumoniae 1 μgmL⁻¹Neg M. amphoriforme 1 μgmL⁻¹ Neg M. fermentans 1 μgmL⁻¹ Neg M faucium 1μgmL⁻¹ Neg M. penetrans 1 μgmL⁻¹ Neg M. pirum 1 μgmL⁻¹ Neg M.spermatophilum 1 μgmL⁻¹ Neg M. primatum 1 μgmL⁻¹ Neg M. salivarium 1μgmL⁻¹ Neg M. buccale 1 μgmL⁻¹ Neg M. orale 1 μgmL⁻¹ Neg M. lipophilum 1μgmL⁻¹ Neg U. parvum (Sero 1) 1 μgmL⁻¹ Neg U. urealyticum (Sero 12) 1μgmL⁻¹ NegSummary—the Assay does not Amplify Other Bacteria or Mollicutes

2) Sensitivity

Standard conc (μgmL⁻¹) Result Cp (average) 100 Pos 15.28, 15.16 (15.22)10 Pos 19.2, 19.07 (19.14) 1 Pos 24.37, 21.37 (24.37) −1 Pos 27.33,27.28 (27.3) −2 Pos 31.27, 31.41, 31.08, 31.12 (31.22) −3 Pos 34.46,35.33, 34.84, 36.27 (35.17) −4 Pos 37.87, 39.17, 38.18, — (38.41) −5 Neg38.56, —, —, — −6 Neg —, —, —, — −7 Neg —, —, —, —

LDL Calculations:

The lowest detectable limit of detection was 5 μL of a 10⁻4 dilution ofa 1 μgmL⁻¹ extract of DNA. This corresponds to 0.0001 μgmL⁻¹ or 100 pg,0.5 pg per 5 μL in reaction and 825 gene copies per reaction. The assaycould detect DNA at one further dilution (at 82.5 gene copies perreaction), however reproducibility was then affected.

The assay LDL is=0.5 pg per reaction

The point at which a real-time PCR is determined as positive can beknown as a crossing point (Cp). A lower Cp indicates a larger amount oftarget DNA in the sample or more efficient amplification. For allpositive specimens to date the mean Cp was 34 in the Mg219 assay of thepresent invention, and 36 in the known MgPa assay.

This indicates that the Mg219 assay of the present invention may be moresensitive that the known MgPa assay.

3) Known Positives/Negatives

Sample Sample Expected Result Actual result type number/ID (Mg219 assay)(MgPa assay) Urethral isolate M30 10-4 P P* (J. Tully) Urethral isolateM 30 early 10-2 P P* (J. Tully) Human throat TW10-5G 10-4 P P* (J.Tully) Human throat TW10-6 G10-4 P P* (J. Tully) Human Throat R-32G 10-3P P* (J. Tully) Human throat TW 48-5G 10-4 P P* (J. Tully) Urethra M2300 10-4 P P* (J. Jensen) Urethra M2321 10-3 P P* (J. Jensen) UrethraM2341 10-3 P P* (J. Jensen) Urethral isolate G37 10-4 P P* (D. T.Robinson) Urethra M 2288 10-3 P P* (J. Jensen) Synovial fluid UTMB-10G10-4 P P* (J. Tully) Water N/A N N PBS N/A N N P = positive in Jensen etal. (2004) publication Summary - all known positives are detected. Knownnegatives are not reactive in the assay

4) Optimal Extraction Method

Extraction of M. hominis spiked urine was performed using varyingmethods (boiling, qiagen, magnapure compact). The magnapure compact gavethe optimal results.

-   -   Samples are best stored at 4° C., not at room temperature.    -   DNA was detectable for up to 28 days.    -   The addition of RND/DNA protect enhances detection and        eliminates the temperature differences—but does not alter        results and is not therefore cost effective.    -   Concentration of sample prior to extraction of DNA is        recommended.

5) Alternative PCR Platforms

The Mg219 assay has been tested on the Roche Lightcycler and the ABITaqman 7700. The assay performed satisfactorily on both platforms(positive controls were amplified and detected whilst negative controlswere not).

These results indicate that the chemistry used in the Mg219 assay istransferable to other real-time platforms.

6) Varying Sample Types (Comparison with MqPa Assay)

Sample Mg219 (actual result) MgPa assay (expected result) Urine 0/112positive 0/112 positive (1 FP) Rectal swabs 2/22 positive 2/22 positiveAnal swab 0/1 positive 0/1 positive Urethral swabs 0/13 positive 0/13positive Genital ulcer 0/18 positive 0/18 positive swabs Penile swab 1/1positive 0/1 positive Liquid based 0/30 positive 0/30 positive cytologysamples - cervical swab Vaginal swabs 0/35 positive 0/35 positiveRespiratory 0/38 positive 0/38 positive (1 FP) samples Endocervical5/309 positive 5/309 positive (7 FP) samples Defined 8/579 positive(1.38%) 7/579 positive (1.21%) (9 FP) samples total FP—false positive -specimens positive only in the MgPa assay are thought to be falsepositive results (see below).

7) Reproducibility

Mg219 controls, CT values when run on several days

Expt Date number 1/10 1/100 1/1000 16/2/6 0236 13.86 17.17 22.8 17/2/60240 12.39 16.89 22.23 24/2/6 0247 11.24 16.63 22.14 02/3/6 0249 10.9917.21 24.01 03/3/6 0251 12.83 19.08 23.84 7/4/6 0255 11.66 17.44 23.2311/5/6 0258 12.84 15.81 19.31 AVERAGE 12.26 17.18 22.51 STDEV  1.02 1.00  1.59 Boundaries 10.22-14.30 15.19-19.17 19.34-25.68 (+/−2 ×STDEV) Note all runs are within average +/−2 × STDEV.

8) Comparison to Known MqPa Assay (Jensen et al., 2004)

Known MgPa assay New HPA MG219 assay Specificity Does not cross reactwith Does not cross react with any any human Mycoplasma human Mycoplasmaspecies species Does not cross react with Does not cross react withbacteria and viruses listed bacteria and viruses listed above above Geneand primers/probes do not align to any other genes with significanthomology on search of BLAST and other public databases Using ORFANAGEprogram MG219 is found only in M. genitalium and not in other publishedwhole genome sequences Using MOLLIGEN whole genome comparison, MG219 isnot found in any other Mycoplasma species. Sensitivity Lower detectionlimit = 0.5 pg Lower detection limit = 0.5 pg per reaction of M.genitalium per reaction of M. genitalium DNA. DNA (same as MgPa assay).Known Detects all known positives Detects all known positives positivestested (culture positive) tested (culture positive) Known Does notamplify known Does not amplify known negatives negatives negativesSample MgPa assay (expected result) Mg219 (actual result) Urine 0/112pos (1FP) 0/112 pos Rectal swabs 2/22 pos 2/22 pos Anal swab 0/1 pos 0/1pos Urethral swabs 0/13 pos 0/13 pos Genital ulcer 0/18 pos 0/18 posswabs Penile swab 0/1 pos 1/1 pos (1FP) LBC samples - 0/30 pos 0/30 poscervical swab Vaginal swabs 0/35 pos 0/35 pos Respiratory 0/38 pos (1FP)0/38 pos samples Endocervical 5/309 pos (7FP) 5/309 pos samples Defined7/579 pos (9FP) 8/579 pos (1FP) samples total Undefined 4/155 pos (11FP)4/155 (0FP) samples (query type)

Example 3 Epidemiological Data (UK Population)

To date, 548 specimens have been tested, of which 9 (1.65%) werepositive in the MG219 assay (see Table below). This indicates a level ofapproximately 1.65% infected individuals within the UK population.

Epidemiological information from research study specimens Known MgPaassay Mg219 (actual result) (expected result) Cornwall NHS 0/134 pos(0%) 0/134 pos (3 FP) Trust University 5/274 pos (1.82%) 5/274 (5 FP)Hospital College London Northampton 4/140 pos (2.86%) 4/140 pos (6 FP)General Hospital Research study 9/548 pos (1.65%) 9/548 pos (14 FP)specimens total

The samples are taken from both symptomatic and asymptomatic patientsand therefore the true level of infection in specific clinical groups(such as those without symptoms or with urethritis) may be lower orhigher than reflected herein. Also, these samples are in the main from 3geographical areas (Cornwall, London, Northampton) with levels varyingfrom 0%, 1.82% to 2.86%. Larger scale screening will indicate ifgeographical differences occur in infection levels.

False Positive Specimens in the Known MgPa Assay:

To date we have 20 specimens which are positive according to the MgPaassay described by Jensen et al. (2004) and negative in the Mg219 assayof the present invention. On repeat testing only 2 of the 20 werepositive in the MgPa test, which indicates that they are likely to befalse positives in the Jensen et al. MgPa test. In this regard, thepresent inventors have been unable to repeat the MgPa positive resultand have not had any positives using two other PCR methods (Cadieux N.et al., (1993) J Gen Microbiol. 1993 Oct.; 139(10): 2431-7, and MG192PCR by Musatovova O. et al., (2006) J Clin Microbiol. 44(2):598-603).

The mean Cp for these samples was 39, which could be indicative ofinefficient amplification due to either lower amounts of target DNA ornon-specific amplification. The former is unlikely, because all samplespositive in both assays had lower mean Cp in the MG219 assay of thepresent invention than in the known MgPa assay.

This indicates that the Mg219 assay of the present invention is moresensitive than the known MgPa assay for the detection of M. genitalium.

Example 4 Further Primer Design A. Designing Primers for PCR of Whole ofMG219

Two primers were designed binding upstream and downstream of the Mg219nucleic acid sequence. As illustrated in the Table below, the up-stream,forward primer SEQ ID NO: 9 mapped to position −76 to −54 (upstream ofthe start codon) and the downstream, reverse primer SEQ ID NO: 20 mappedto position 503 to 525 (downstream of the stop codon) of MG219. Theannealing temperature of the primers was calculated using the formula:Tm=4*(number of G's and C's)+2*(number of A's and T's).

Primer Sequence Position SEQ ID NO: 9 CCACTTAACTTTATTACCCGTCC −76 to −54SEQ ID NO: 20 GATTAACCCCAGGTAGTTCTTCC +503-525

To confirm the suitability of the primers, BLAST analysis was carriedout for each of the primers against the M. genitalium G37 genome.Selecting the “Advanced Blast” parameters, small primer sequences showedunique binding sites and enabled determination of homologous sequenceselsewhere on the genome, and prediction of possible multipleamplification products upon PCR.

B. Determining Optimum Annealing Temperature for PCR

To optimise the annealing temperature and avoid multiple PCR products,gradient PCR was performed using the primers SEQ ID NO: 9 and SEQ ID NO:20, ranging from 57-68° C. This was performed on a MJ Research DNAengine, which does not perform a linear gradient across the block (FIG.3).

No other products were observed indicating that only the MG219 gene wasamplified. The resulting amplicons were used to determine the sequenceof the MG219 gene and to amplify across the gene.

C. Detection of Mg219

Forward primers SEQ ID NOs: 10-19 were designed starting from position−53 relative to the start codon and mapping consecutively to 3 basesbeyond the stop codon, to cover the entire gene sequence of Mg219 (seeTable below).

Forward Primer SEQ ID NOs: Sequence Position 10TGTTTTCAAAAGTAATTTGCCACCGAAACTAA −53 to −1 GTAAGGATGACATAGTTCATT 11ATGCGCACCAGTTACTTGAAAAAAATACCCAT  1-50 AATGAATAGTGATAGTGA 12TCTAAAACTCCAAAAGGTGTGGATCGAGCGGC  51-100 ATGTTGATCAAGATGAAC 13TTAGTTTAACAACTACTGCAGTTGAACTTAAA 101-150 AAGAGTGATGAACAAAAA 14CCTGTTGCCATTAAAAGTAGTGACTTTATTGG 151-200 TCATGAAGAGTTAATCTC 15TGTTCCAGTTTTACTAATCCCAACCCCTGTTG 201-250 TTAAAGAGATTGATCAAC 16CAGCAGTTATTCCTCCAGTTAAAGCAAAACCA 251-300 AAAGCAACTAAAAAGAAA 17ACTCCTGTTAAATCAAAACCAACTAGTAAATC 301-350 AACTAAACAAACAAAACC 18TAAACAATCCAAGCCCAAATCAAAACAAGTTC 351-400 AACAAACCAAAGCTAAAC 19CAACCCAAATTCAAACAAAAAAAAGCAATAAA  401-+450 AAAACCAGATCTTAATCT

Reverse primer SEQ ID NOs: 21-30 were designed starting from position+502 from the start codon and they map consecutively upstream towardsthe start codon (see Table below).

Reverse Primer SEQ ID NOs: Sequence Position 21TATTCCTTTCCAGTTTTTAGTTAAAACTACTG +448-+502 TTGTTAACACTAAAAAAACCAGA 22TTAAGATCTGGTTTTTTTATTGCTTTTTTTTG 398-447 TTTGAATTTGGGTTGGTT 23TAGCTTTGGTTTGTTGAACTTGTTTTGATTTG 348-397 GGCTTGGATTGTTTAGGT 24TTTGTTTGTTTAGTTGATTTACTAGTTGGTTT 298-347 TGATTTAACAGGAGTTTT 25CTTTTTAGTTGCTTTTGGTTTTGCTTTAACTG 248-297 GAGGAATAACTGCTGGTT 26GATCAATCTCTTTAACAACAGGGGTTGGGATT 198-247 AGTAAAACTGGAACAGAG 27ATTAACTCTTCATGACCAATAAAGTCACTACT 148-197 TTTAATGGCAACAGGTTT 28TTGTTCATCACTCTTTTTAAGTTCAACTGCAG  98-147 TAGTTGTTAAACTAAGTT 29CATCTTGATCAACATGCCGCTCGATCCACACC 48-97 TTTTGGAGTTTTAGATCA 30CTATCACTATTCATTATGGGTATTTTTTTCAA −3-47 GTAACTGGTGCGCATAAT

Individual PCRs were performed using forward primer SEQ ID NO: 9 plusany one of reverse primers SEQ ID NOs: 21-30; and also using forwardprimer SEQ ID NO: 20 plus any one of reverse primers SEQ ID NOs: 10-19.

As illustrated in FIGS. 4 and 5, PCR amplification using the primerpairs described above detected the whole Mg219 gene.

Example 5 Conservation of the Mg219 Gene

PCR amplification of the entire Mg219 gene and flanking regions(upstream and downstream sequences) was performed using primers SEQ IDNO: 9 and SEQ ID NO: 20 on twelve cultured M. genitalium isolates (seeTable below) and the type strain MG37 using Roche High Fidelity Taq.

The resultant amplified fragments were all of the same size (exceptisolates 9 and 11, which appeared slightly larger on agarose gelelectrophoresis) see FIG. 6.

Cultured Isolate M. genitalium Number strain name Origin 1 M30 10-4Urethra 2 M30 early 10-2 Urethra 3 TW10-5G 10-4 Throat 4 TW10-6G 10-4Throat 5 R-32G 10-3 Throat 6 TW48-5G 10-4 Throat 7 M2300 10-4 Urethra 8M2321 10-3 Urethra 9 M2341 10-3 Urethra 10 G37 10-4 Urethra 11 M228810-3 Urethra 12 UTMB 10G 10-4 Synovial fluid G37 NCTC type strainUrethra

The resulting amplified fragments were purified and the concentrationsdetermined. Both strands of the PCR products were sequenced using theoligonucleotide primers SEQ ID NO: 9 and SEQ ID NO: 20 in triplicate.Sequencing was performed using the Becton-Dickinson sequencing kitfollowing the manufacture's instructions and using the CEQ8000 DNAsequencer instrument and the consensus sequence for each strain wasdetermined using MapVector and ClustalW software.

Sequences were aligned and the alignment showed high conservation of theMG219 gene (see Table below and FIGS. 7 and 8). Only 40 bases differedfrom the 569 bases of the control G37 strain and these differences werefound only within strains 9 and 11. Of these, 30 bases consisted of aninsertion in isolates 9 and 11 between residues 504 and 505 of thecontrol strain.

Further subsequent sequence analysis of four positive samples(identified as +ve MG 2, +ve MG 3, +ve MG 5 and +ve MG 6, below) hasdetermined that positive clinical specimens are also highly conserved.The insertion sequence is also present in the sequence from Sample +veMG 6.

MG219 identity Isolate Number SEQ ID NO: to G37 strain 1 32 100% 2 33100% 3 34 100% 4 35 100% 5 36 100% 6 37 100% 7 38 100% 8 39 100% 9 4093.1% (40/569) 10 41 100% 11 42 93.2% (39/569) 12 43 100% +ve MG 2 4499.8% (1/569)  +ve MG 3 45 100% +ve MG 5 46 100% +ve MG 6 47 93.2%(39/569) G37 48 N/A

1. A method for detecting M. genitalium nucleic acid in a sample,comprising: (i) amplifying a nucleic acid sequence comprising a fragmentof SEQ ID NO: 1; and (ii) detecting said amplified nucleic acidsequence.
 2. A method according to claim 1, wherein said amplifying stepcomprises contacting said sample with forward and reverseoligonucleotide primers, wherein said primers bind to target sites inthe M. genitalium nucleic acid, under conditions suitable to promoteamplification of said nucleic acid sequence comprising a fragment of SEQID NO:
 1. 3. A method according to claim 2, wherein the forward primerbinds to a target site located between nucleotides −80 to 450 of anucleic acid strand complementary to SEQ ID NO: 1, preferably locatedfrom nucleotide residue −76 of a nucleic acid strand complementary toSEQ ID NO: 1 (for example located from nucleotide residue −60, −55, −53,−50, −25, −10, 1, 25, 51, 101, 151, 201, 251, 301, 351 or 401 of anucleic acid strand complementary to SEQ ID NO: 1), and preferablylocated up to nucleotide residue 447 of a nucleic acid strandcomplementary to SEQ ID NO: 1 (for example located up to nucleotideresidue 450, 425, 400, 350, 300, 250, 200, 150, 100, 50, 25, 1, −5, −10,−25, −50, −54 or −55 of a nucleic acid strand complementary to SEQ IDNO: 1).
 4. A method according to claim 3, wherein the forward primerbinds to a target site located between nucleotide residues −25 to 25 ofa nucleic acid strand complementary to SEQ ID NO: 1, preferably to atarget site defined by residues −11 to 18 of a nucleic acid strandcomplementary to SEQ ID NO:
 1. 5. A method according to any of claims 2to 4, wherein the reverse primer binds to a target site located betweennucleotide residues −5 to +525 of SEQ ID NO: 1, preferably located fromnucleotide residue −3 of SEQ ID NO: 1 (for example located fromnucleotide residue 1, 10, 25, 48, 98, 148, 198, 248, 298, 348, 398, +448or +503 of SEQ ID NO: 1), and preferably located up to nucleotideresidue +502 of SEQ ID NO: 1 (for example located up to nucleotideresidue 447, 397, 347, 297, 247, 197, 147, 97, 47 or 25 of SEQ ID NO:1).
 6. A method according to claim 5, wherein the reverse primer bindsto a target site located between nucleotide residues 200 to 250 of SEQID NO: 1, and preferably to a target site defined by nucleotide residues214 to 240 of SEQ ID NO:
 1. 7. A method according to any of claims 2 to6 wherein said forward and reverse oligonucleotide primers are at least5 nucleotides long, preferably at least 10 nucleotides long, morepreferably at least 20 nucleotides long, and preferably no more than 50nucleotides long, more preferably no more than 40 nucleotides long, mostpreferably no more than 30 nucleotides long.
 8. A method according toclaim 7, wherein said forward primer comprises a nucleic acid sequenceselected from SEQ ID NOs: 4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 31, or a fragment thereof, or a variant thereof having at least60% sequence identity thereto.
 9. A method according to claim 7 or 8,wherein said reverse primer comprises a nucleic acid sequence selectedfrom SEQ ID NOs: 5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or afragment thereof, or a variant thereof having at least 60% sequenceidentity thereto.
 10. A method according to any previous claim, whereinsaid amplified nucleic acid sequence is detected by gel electrophoresis.11. A method according to any of claims 1-10, wherein said detectingstep comprises: (i) contacting said amplified nucleic acid sequence witha probe, wherein the probe binds to a target site within said amplifiednucleic acid sequence, or the complement thereof; and (ii) detectingbinding of said probe to said amplified nucleic acid sequence.
 12. Amethod for detecting M. genitalium nucleic acid in a sample, comprising:(i) contacting said sample with a probe, wherein the probe binds to atarget site within SEQ ID NO: 1, or the complement thereof; and (ii)detecting binding of said probe to said target site.
 13. A methodaccording to claim 11 or 12, wherein said probe binds to a target sitelocated between nucleotide residues 1-447 of SEQ ID NO: 1, or thecomplement thereof, preferably located from nucleotide residue 10 of SEQID NO: 1 or the complement thereof (for example from nucleotide residue15, 25, 50, 75, 100, 150, 200, 250, 300, 350 or 400 of SEQ ID NO: 1 orthe complement thereof) and preferably located up to nucleotide residue440 of SEQ ID NO: 1, or the complement thereof (for example fromnucleotide residue 420, 400, 350, 300, 250, 200, 150, 100 or 50 of SEQID NO: 1 or the complement thereof).
 14. A method according to claim 13,wherein the probe binds to a target site located between nucleotideresidues 50-100 of the nucleic acid strand complementary to SEQ ID NO:1, and preferably to a target site defined by nucleotides 66-82 of thenucleic acid strand complementary to SEQ ID NO:
 1. 15. A methodaccording to any of claims 11-14 wherein the probe is an oligonucleotideprobe.
 16. A method according to claim 15 wherein said probe is at least5 nucleotides long, preferably at least 10 nucleotides long, morepreferably at least 15 nucleotides long, most preferably at least 20nucleotides long, and wherein said probe is up to 50 nucleotides long,preferably up to 40 nucleotides long, more preferably up to 30nucleotides long.
 17. A method according to claim 16 wherein the probecomprises a nucleic acid sequence of SEQ ID NO: 3, or a fragmentthereof, or a variant thereof having at least 60% sequence identitythereto.
 18. A method according to claim 17, wherein the probe consistsof a nucleic acid sequence of SEQ ID NO: 3, or a fragment thereof, or avariant thereof having at least 60% sequence identity thereto.
 19. Amethod according to any of claims 11-18, wherein said probe isimmobilised onto a solid support or platform.
 20. A method according toany of claims 11-19, wherein binding of said probe provides a detectablesignal, and wherein said detecting step comprises detecting said signal.21. A method according to claim 20, wherein said detectable signal is afluorescent signal.
 22. A method according to claim 21, wherein theprobe is labelled.
 23. A method according to any previous claim, whereinthe sample comprises human semen and/or human eggs; and wherein thesubstantial absence of said signal indicates that the sample issubstantially free of M. genitalium nucleic acid, and that the humansemen and/or eggs can be used for in vitro fertilisation.
 24. An invitro method for quantitating M. genitalium pathogen load in a sample ofinterest, comprising: (a) carrying out a method according to any ofclaims 1-22 on said sample of interest; and (b) carrying out a methodaccording to any of claims 1-22 on a test sample of predetermined knownM. genitalium pathogen load; and (c) comparing the signal detected fromthe sample of interest with the signal detected from the test sample;and thereby quantitating M. genitalium pathogen load in the sample ofinterest.
 25. An in vitro method for determining drug efficacy over thecourse of a period of drug therapy, comprising: (a) carrying out amethod according to any of claims 1-22 on a first sample obtained at afirst time point within or prior to the period of drug therapy; (b)carrying out a method according to any of claims 1-22 on a sample at oneor more later time points within the period of drug therapy; and (c)comparing the signal detected from the first sample with the signaldetected from the one or more later samples; and thereby determiningdrug efficacy over the course of the period of drug therapy.
 26. An invitro method for detecting and/or monitoring the development ofresistance of M. genitalium to a drug, comprising: (a) carrying out amethod according to any of claims 1-22 on a first sample obtained at afirst time point within or prior to exposure of the M. genitalium to thedrug; (b) carrying out a method according to any of claims 1-22 on asample at one or more later time points following exposure of the M.genitalium to the drug; and (c) comparing the signal detected from thefirst sample with the signal detected from the one or more latersamples; wherein the absence of a reduction in the signal, or anincrease in the signal, indicates that the M. genitalium have developedresistance to the drug.
 27. A probe for use in a method according to anyprevious claim, wherein said probe binds to a target sequence withinsaid amplified nucleic acid sequence, or the complement thereof, orwithin SEQ ID NO: 1, or the complement thereof.
 28. A probe according toclaim 27, wherein said probe binds to a target site located betweennucleotide residues 1-447 of SEQ ID NO: 1, or the complement thereof,preferably located from nucleotide residue 10 of SEQ ID NO: 1 or thecomplement thereof (for example from nucleotide residue 15, 25, 50, 75,100, 150, 200, 250, 300, 350 or 400 of SEQ ID NO: 1 or the complementthereof) and preferably located up to nucleotide residue 440 of SEQ IDNO: 1, or the complement thereof (for example from nucleotide residue420, 400, 350, 300, 250, 200, 150, 100 or 50 of SEQ ID NO: 1 or thecomplement thereof).
 29. A probe according to claim 28, wherein theprobe binds to a target site located between nucleotide residues 50-100of the nucleic acid strand complementary to SEQ ID NO: 1, and preferablyto a target site defined by nucleotides 66-82 of the nucleic acid strandcomplementary to SEQ ID NO:
 1. 30. A probe according to any of claims27-29, wherein the probe is an oligonucleotide probe.
 31. A probeaccording to claim 30 wherein said probe is at least 5 nucleotides long,preferably at least 10 nucleotides long, more preferably at least 15nucleotides long, and wherein said probe is up to 50 nucleotides long,preferably up to 40 nucleotides long, more preferably up to 30nucleotides long, more preferably up to 25 nucleotides long, mostpreferably up to 20 nucleotides long.
 32. A probe according to claim 30or 31, wherein the probe comprises a nucleic acid sequence of SEQ ID NO:3, or a fragment thereof, or a variant thereof having at least 60%sequence identity thereto.
 33. A probe according to claim 32, whereinthe probe consists of a nucleic acid sequence or SEQ ID NO: 3, or afragment thereof, or a variant thereof having at least 60% sequenceidentity thereto.
 34. A probe according to any of claims 27-33, whereinsaid probe provides a detectable signal on binding to said targetsequence.
 35. A probe according to claim 34, wherein said detectablesignal is a fluorescent signal.
 36. A probe according to claim 35,wherein the probe is labelled.
 37. A forward primer for use in a methodaccording to any previous claims, wherein said forward primer binds to atarget site located either (i) within the complement of SEQ ID NO: 1,(ii) within upstream nucleic acid sequences that flank the complement ofSEQ ID NO: 1, or (iii) overlapping the complement of SEQ ID NO: 1 andupstream nucleic acid sequences that flank the complement of SEQ IDNO:
 1. 38. A forward primer according to claim 37, wherein the forwardprimer binds to a target site located between nucleotides −80 to +450 ofa nucleic acid strand complementary to SEQ ID NO: 1, preferably locatedfrom nucleotide residue −76 of a nucleic acid strand complementary toSEQ ID NO: 1 (for example located from nucleotide residue −60, −55, −53,−50, −25, −10, 1, 25, 51, 101, 151, 201, 251, 301, 351 or 401 of anucleic acid strand complementary to SEQ ID NO: 1), and preferablylocated up to nucleotide residue 447 of a nucleic acid strandcomplementary to SEQ ID NO: 1 (for example located up to nucleotideresidue 425, 400, 350, 300, 250, 200, 150, 100, 50, 25, 1, −5, −10, −25,−50, −54 or −55 of a nucleic acid strand complementary to SEQ ID NO: 1).39. A forward primer according to claim 38, wherein the forward primerbinds to a target site located between nucleotide residues −25 to 25 ofa nucleic acid strand complementary to SEQ ID NO: 1, preferably to atarget site defined by residues −11 to 18 of a nucleic acid strandcomplementary to SEQ ID NO:
 1. 40. A forward primer according to any ofclaims 37 to 39, wherein said primer is at least 5 nucleotides long,preferably at least 10 nucleotides long, more preferably at least 15nucleotides long, more preferably at least 20 nucleotides long, mostpreferably at least 25 nucleotides long, and wherein said probe is up to50 nucleotides long, preferably up to 40 nucleotides long, morepreferably up to 30 nucleotides long.
 41. A forward primer according toany of claims 37 to 39, wherein said forward primer comprises a nucleicacid sequence selected from SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 31, or a fragment thereof, or a variant thereof havingat least 60% sequence identity thereto.
 42. A reverse primer for use ina method according to any previous claim, wherein said reverseoligonucleotide primer binds to a target site located either (i) withinSEQ ID NO: 1, (ii) within downstream nucleic acid sequences that flankSEQ ID NO: 1, or (iii) overlapping SEQ ID NO: 1 and downstream nucleicacid sequences that flank SEQ ID NO:
 1. 43. A reverse primer accordingto claim 42, wherein the reverse primer binds to a target site locatedbetween nucleotide residues −5 to +525 of SEQ ID NO: 1, preferablylocated from nucleotide residue −3 of SEQ ID NO: 1 (for example locatedfrom nucleotide residue 10, 25, 48, 98, 148, 198, 248, 298, 348, 398,+448 or +503 of SEQ ID NO: 1), and preferably located up to nucleotideresidue +502 of SEQ ID NO: 1 (for example located up to nucleotideresidue 447, 397, 347, 297, 247, 197, 147, 97, 47 or 25 of SEQ ID NO:1).
 44. A reverse primer according to claim 43, wherein the reverseprimer binds to a target site located between nucleotide residues 200 to250 of SEQ ID NO: 1, and preferably to a target site defined bynucleotide residues 214 to 240 of SEQ ID NO:
 1. 45. A reverse primeraccording to any of claims 42-44, wherein said primer is at least 5nucleotides long, preferably at least 10 nucleotides long, morepreferably at least 15 nucleotides long, more preferably at least 20nucleotides long, most preferably at least 25 nucleotides long, andwherein said probe is up to 50 nucleotides long, preferably up to 40nucleotides long, more preferably up to 30 nucleotides long.
 46. Areverse primer according to any of claims 42-45, wherein said reverseprimer comprises a nucleic acid sequence selected from SEQ ID NO: 5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or a fragment thereof, or avariant thereof having at least 60% sequence identity thereto.
 47. Apair of oligonucleotide primers, comprising a forward primer accordingto any of claims 37-41 and a reverse primer according to any of claims42-46.
 48. A kit for detection of M. genitalium nucleic acid comprisinga probe according to any of claims 27-36.
 49. A kit according to claim48, further comprising a forward primer according to any of claims37-41.
 50. A kit according to claim 48 or 49, further comprising areverse primer according to any of claims 42-46.
 51. A method ofdetecting M. genitalium nucleic acid in a sample, substantially asherein described, with reference to the Examples and/or to FIG.
 1. 52. Aprobe substantially as herein described, with reference to FIG.
 1. 53. Aforward primer substantially as herein described, with reference to FIG.1 or
 2. 54. A reverse primer substantially as herein described, withreference to FIG. 1 or
 2. 55. A kit substantially as herein described,with reference to FIG. 1 or 2.